dCas9-TET1 conditional knock-in mice carry a loxP-flanked STOP cassette preventing widespread expression of the RNA-guided DNA-binding fusion protein dCas9-TET1 - a codon-optimized, nuclease-deficient, catalytically dead SpCas9 protein (dCas9) fused to the catalytic domain of human tet methylcytosine dioxygenase 1 (TET1). Following Cre recombinase exposure and introduction of a single guide RNA (sgRNA) that directs dCas9-TET1 binding to a specific DNA sequence/locus, the TET1 domain subsequently catalyzes demethylation of target sites. These mice allow Cre-inducible, RNA-guided DNA demethylation of target genes. More specific details are below. The dCas9-TET1 knock-in allele has a CAG promoter and loxP-flanked STOP cassette upstream of the dCas9-TET1 fusion gene - all inserted into the Gt(ROSA)26Sor locus. Prior to the introduction of Cre recombinase, mice heterozygous or homozygous for the dCas9-TET1 allele are viable and fertile with normal fertility, and no gross physical or behavioral abnormalities. The donating investigator reports that no dCas9-TET1 expression is observed without Cre exposure by Western blot and GFP signal. Upon exposure to Cre recombinase, the STOP cassette is removed - resulting in dCas9-TET1 expression in the cells/tissues as determined by the Cre promoter. Specifically, the donating investigator reports that when using dCas9-TET1 with Cre recombinase expression directed to early embryonic tissues, brain, and liver, the resulting mice all show the desired dCas9-TET1 expression levels, with appropriate target gene demethylation as determined by the sgRNA used. Through Cre-dependent control, in conjunction with the use of dCas9-DNMT3A mice (Stock No. <a href="https://www.jax.org/strain/041503">041503</a>), these models enable precise spatial and temporal modulation of the targeted manipulation of DNA methylation/demethylation.

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dCas9-TET1 conditional knock-in mice carry a Cre-inducible dCas9-TET1 fusion protein - a codon-optimized, nuclease-deficient, catalytically dead SpCas9 protein (dCas9) fused to the catalytic domain of the human tet methylcytosine dioxygenase 1 (TET1) inserted into the Rosa26 locus. Following Cre recombinase and introduction of a single guide RNA (sgRNA), this model enables precise spatial and temporal DNA demethylation of the genome.

Heterozygous for Gt(ROSA)26Sor<tm2(CAG-dcas9/TET1,-EGFP)Xsl>

Homozygous for Gt(ROSA)26Sor<tm2(CAG-dcas9/TET1,-EGFP)Xsl>

Wildtype for Gt(ROSA)26Sor<tm2(CAG-dcas9/TET1,-EGFP)Xsl>